Article Fungal Community and Ligninolytic Enzyme Activities in Quercus deserticola Trel. Litter from Forest Fragments with Increasing Levels of Disturbance

Jesús A. Rosales-Castillo 1,2, Ken Oyama 1, Ma. Soledad Vázquez-Garcidueñas 3, Rafael Aguilar-Romero 1, Felipe García-Oliva 4 and Gerardo Vázquez-Marrufo 2,* 1 Escuela Nacional de Estudios Superiores Unidad Morelia, Universidad Nacional Autónoma de México (UNAM), Antigua Carretera a Pátzcuaro No. 8701, Colonia Ex Hacienda de San José de La Huerta, Morelia 58190, Michoacán, Mexico; [email protected] (J.A.R.-C.); [email protected] (K.O.); [email protected] (R.A.-R.) 2 Centro Multidisciplinario de Estudios en Biotecnología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Michoacana de San Nicolás de Hidalgo, Km 9.5 carretera Morelia-Zinapécuaro, Col. La Palma Tarímbaro, Morelia 58893, Michoacán, Mexico 3 División de Estudios de Posgrado, Facultad de Ciencias Médicas y Biológicas “Dr. Ignacio Chávez”, Universidad Michoacana de San Nicolás de Hidalgo, Ave. Rafael Carrillo esq. Dr. Salvador González Herrejón. Col. Cuauhtémoc, Morelia 58020, Michoacán, Mexico; [email protected] 4 Instituto de Investigaciones en Ecosistemas y Sustentabilidad, Universidad Nacional Autónoma de México (UNAM), Antigua Carretera a Pátzcuaro No. 8701, Colonia Ex Hacienda de San José de La Huerta, Morelia 58190, Michoacán, Mexico; [email protected] * Correspondence: [email protected]; Tel./Fax: +52-443-295-8029 (ext. 2)

Received: 3 October 2017; Accepted: 21 December 2017; Published: 23 December 2017

Abstract: Litter fungal communities and their ligninolytic enzyme activities (laccase, Mn-peroxidase, and lignin-peroxidase) play a vital role in forest biogeochemical cycles by breaking down plant cell wall polymers, including recalcitrant lignin. However, litter fungal communities and ligninolytic enzyme activities have rarely been studied in Neotropical, non-coniferous forests. Here, we found no significant differences in litter ligninolytic enzyme activities from well preserved, moderately disturbed, and heavily disturbed Quercus deserticola Trel. forests in central Mexico. However, we did find seasonal effects on enzyme activities: during the dry season, we observed lower laccase, and increased Mn-peroxidase and lignin-peroxidase activities, and in the rainy season, Mn-peroxidase and lignin-peroxidase activities were lower, while laccase activity peaked. Fungal diversity (Shannon-Weaver and Simpson indices) based on ITS-rDNA analyses decreased with increased disturbance, and principal component analysis showed that litter fungal communities are structured differently between forest types. White-rot Polyporales and Auriculariales only occurred in the well preserved forest, and a high number of Ascomycota were shared between forests. While the degree of forest disturbance significantly affected the litter fungal community structure, the ligninolytic enzyme activities remained unaffected, suggesting functional redundancy and a possible role of generalist Ascomycota taxa in litter delignification. Forest conservation and restoration strategies must account for leaf litter and its associated fungal community.

Keywords: oak litter; ligninolytic enzymes; forest litter degradation; fungal community

1. Introduction Litter is a key component of nutrient dynamics in forest ecosystems that, upon its decomposition, provides available nutrients for the plant community [1]. Litter decay in forest ecosystems is a dynamic

Forests 2018, 9, 11; doi:10.3390/f9010011 www.mdpi.com/journal/forests Forests 2018, 9, 11 2 of 18 process involving the participation of soil fauna and a complex microbial community producing extracellular lignocellulolytic enzymes [2]. Within litter biota, the fungal community performs roles in polysaccharide and lignin degradation [3], causing changes both in biotic and abiotic factors at different spatial and temporal scales [4]. The structure and function of fungal communities in the forest floor have patterns characteristic to forest types, with well-established differences between forests in temperate and tropical ecosystems [5] and between forests dominated by coniferous and broadleaf trees [6,7]. In addition, significant differences have been clearly documented among similar forest types along geographical gradients [8]. Furthermore, it has been noted that the principle significant difference in fungal communities in the same ecosystem generally occurs between communities in the litter and in the soil, rather than between the organic and mineral soil horizons [8]. Taking all this into account, the structure and dynamics of litter fungal communities in oak forests in tropical and Neotropical areas are not well understood, and, despite the key role of fungi in the process of litter decay in these ecosystems, knowledge regarding fungal community distribution, abundance, and spatial and temporal changes remains scarce. The richness of oaks (Quercus L.; Fagaceae) has been estimated to be between 450 and 600 species worldwide [9,10], of which 160 to 165 are found in Mexico, making the country a hotspot for oak species [11,12]. Thus, at least 27% of the global species richness of Quercus is found in Mexico, across 5.5% of its territory, in ecosystems ranging from temperate humid to warm and arid climates [13]. Rural communities in Mexico exploit oak and oak-pine forests—mainly for the extraction of firewood and charcoal [14,15]—and some of these forests have also been converted to cropland and grazing areas [16]. As a result, the cover of oak forests in Mexico has been seriously reduced with additional impacts upon their structure and dynamics [17,18]. In this regard, García-Oliva et al. [19] reported that in oak-pine forests in central Mexico, uncontrolled wood extraction significantly reduced the carbon pools and disrupted soil nutrient dynamics. For these reasons, knowledge about how these forests respond to disturbance could contribute to developing strategies for their conservation and sustainable management [20,21], in particular changes in microbiological, biochemical, and nutritional components of oak forest litter due to anthropogenic disturbance [22,23], and their potential for recovery. Although Mexico is a hotspot of Quercus species diversity, studies of fungal communities in oak forests are limited [24], focusing on ectomycorrhizal species [25] but not other functional groups. Thus, evaluating the structure of the fungal community and the enzymatic profiles of lignin degradation in decomposing litter allows the characterization of spatial and temporal patterns of diversity and responses to environmental factors [23,26] and land use practices [27,28]. In turn, increased knowledge of forest soil and litter fungal communities will aid in identifying the consequences of changes in microbial diversity on ecosystem functions and services [29], as well as establishing restoration [30] and management [31] strategies. This study aims to describe the ligninolytic enzyme activities and fungal communities present in Quercus deserticola Trel. litter in forests under different degrees of anthropogenic disturbance, and to evaluate differences associated with the dry and rainy seasons. Previous studies in Quercus spp. dominated ecosystems show that fragmentation significantly reduced the diversity of fungal communities, affecting their metabolic profiles [32]. Based on this and on the above cited works, we predicted that the Q. deserticola litter fungal communities would be less diverse in sites with increasing disturbance and that, independently of the degree of disturbance, at the end of the rainy season, the saprobic guild within basidiomycetes would predominate over mycorrhizal groups. Corresponding changes in ligninolytic enzyme activity of the fungal community may be observed, due to shifts in Basidiomycota functional guilds. Functional consequences of the observed patterns are discussed in the context of ecosystem use at the study site. Forests 2018, 9, 11 3 of 18

2. Materials and Methods

2.1. StudyForests Area2018, 9 and, 11 Litter Sampling 3 of 18

Forest2. Materials fragments and Methods studied are located in the Cuitzeo basin (19◦320 N, 101◦180 W), southeast of the city of Morelia in the Mexican state of Michoacán. The mean annual temperature in the study area is 15.52.1.◦C Study and Area the meanand Litter annual Sampling precipitation is 1047 mm. The July-September period is the wettest period andForest the hottestfragments period studied is April–Juneare located in (http://smn.cna.gob.mx/ the Cuitzeo basin (19°32′ N,). 101°18Quercus′ W), deserticola southeast ofTrel. the trees are dominantcity of Morelia in the in study the Mexican area; they state lose of Michoacán. their foliage The during mean annual the dry temperature season from in the January study toarea May is and flush15.5 leaves °C inand June the atmean the startannual of theprecipitation rainy season is 1047 [24 ,mm.33]. TheThe regionJuly-September in which period the study is the area wettest is located has sufferedperiod and forest the losseshottest dueperiod to is logging April–June for timber(http://smn.cna.gob.mx/). and charcoal extraction, Quercus deserticola agricultural Trel. expansion, trees are dominant in the study area; they lose their foliage during the dry season from January to May and grazing [34–36]. and flush leaves in June at the start of the rainy season [24,33]. The region in which the study area is Within the Cuitzeo basin, three sites with different levels of disturbance were selected: located has suffered forest losses due to logging for timber and charcoal extraction, agricultural well preservedexpansion, and (WP), grazing moderately [34–36]. disturbed (MD), and heavily disturbed (HD) (Table1). In order to minimizeWithin differences the Cuitzeo in geography basin, three and sites climate with diff betweenerent levels sites, of forest disturbance fragments were were selected: located well within 0.5 kmpreserved of each other(WP), andmoderately the dominant disturbed soil (MD), type and in all he threeavily sitesdisturbed was Acrisol.(HD) (Table Because 1). In theorder disturbed to sites showedminimize evidencedifferences of in tree geography extraction and andclimate no signsbetween of burning,sites, forest the fragments conservation were located level ofwithin the three selected0.5 km forest of each fragments other and was the dominant determined soil bytype a in description all three sites of was the Acrisol. vegetation Because structure the disturbed and cover presentsites in showed each site evidence [37] using of tree the extraction method and of no Gentry signs of [38 burning,]. Additionally, the conservation 100 m level× 20 of mthe plots three were establishedselected in forest each fragments of the three was forest determined fragments by a and description the diameters of the vegetation at breast heightstructure (DBH) and cover≥ 5 cm of present in each site [37] using the method of Gentry [38]. Additionally, 100 m × 20 m plots were all individual trees within the plots were measured (Table1). Finally, aboveground biomass (crown established in each of the three forest fragments and the diameters at breast height (DBH) ≥ 5 cm of and stem),all individual litter biomass trees within (Table the1), plots and were plant measured species composition(Table 1). Finally, (Supplementary aboveground Tablebiomass S1) (crown were also usedand as indicators stem), litter of biomass disturbance/regeneration (Table 1), and plant specie in eachs composition plot. Theaboveground (Supplementary biomass Table 1) was were quantified also fromused the DBHas indicators values of using disturbance/regeneration allometric equations in each proposed plot. The by aboveground Aguilar et al.biomass [15]. was The quantified temperatures of thefrom studied the DBH plots values (Table using1) were allometric measured equations with prop a VWRosed by Traceable Aguilar et Logger-Trac al. [15]. The temperatures RH/Temperature of Dataloggerthe studied (Radnor, plots PA,(Table USA). 1) were measured with a VWR Traceable Logger-Trac RH/Temperature Datalogger (Radnor, PA, USA). Table 1. Characteristics of the three forest sites with increasing levels of disturbance in central Mexico studiedTable to examine1. Characteristics fungal of communities the three forest and sites ligninolytic with increasing enzyme levels activities. of disturbance in central Mexico studied to examine fungal communities and ligninolytic enzyme activities.

Well Preserved Moderately Disturbed Heavily Disturbed (WP) (MD) (HD) Coordinates 19°32′13.20″ N, 19°32′18.24″ N, 19°32′6.00″ N, 101°17′60.00″ W 101°17′56.40″ W 101°18′3.60″ W Stand characteristics Number of Quercus deserticola Trel. trees 171 154 39 Mean tree DBH ± standard error (cm) 12.1 ± 0.3 12.3 ± 0.25 15.1 ± 0.6 Aboveground biomass (Mg ha−1) 42.7 46.3 27.4 Mean litter mass ± standard error (Mg ha−1) 1.5 ± 0.25 1.0 ± 0.15 1.1 ± 0.1 Temperature on sampling dates (°C) 1 34.1, 25.6 34.3, 26.1 35, 26.4 Nutrient concentrations Litter 2 pH 5.9–6.1 6.0–6.3 6.1–6.2 Carbon (mg g−1) 417 391 473 Nitrogen (mg g−1) 10.3 8.9 10.4 Phosphorus (mg g−1) 0.34 0.42 0.54 C:N 40 44 45 C:P 1227 935 876 N:P 30 21 19 Soil 2 Carbon (mg g−1) 42.2 58.5 51.3 Nitrogen (mg g−1) 3.4 2.33 2.5 Phosphorus (mg g−1) 0.36 0.17 0.55 C:N 12 25 20 C:P 132 344 93 N:P 11 14 4 1 Temperature1 Temperature data for data the dryfor (June)the dry and (June) rainy and (September) rainy (September) seasons separated seasons by separated a comma. by2 Concentrations a comma. of nutrients2. Concentrations were measured of nutrients in a mixed were sample measured from in 20 a soil mixed subsamples sample from and 20 five soil litter subsamples subsamples and asfive described litter in Materialssubsamples and Methods. as described in Materials and Methods.

Forests 2018, 9, 11 4 of 18

Four transects of 1 m × 100 m of length were established within each plot to quantify nutrient and carbon contents in both the surface litter and the soil beneath it. Along each transect, a soil sample was taken from the top 20 cm with a soil-corer every 20 m. The 20 soil samples were then mixed and kept in a plastic bag. Five samples of litter were randomly collected from each plot within a polyvinyl chloride ring with a diameter of 160 mm. Soil and litter samples were transported to the laboratory in a cooler and placed in darkness at 4 ◦C until analysis [39]. For enzyme activities and fungal community composition, litter sampling was done directly beneath the crown of Q. deserticola trees to avoid heterogeneous litter composition in the degraded sites. In each plot, five oak trees were randomly selected and litter samples were collected in June 2015 (recent leaf-fall) and September 2015 using a 16 cm-diameter polyvinyl chloride ring, including the first 5–10 cm depth to avoid mineral soil layers. The two sampling dates allowed a comparison of the enzyme activity and fungal community between the driest (June) and the wettest (September) seasons of the year. Samples were immediately cooled to 4 ◦C and transported to the laboratory on the same day as collection. Once in the laboratory, each sample was subdivided in two: one subsample was stored at 4 ◦C for physicochemical analyses and enzyme assays, and the other was stored at −80 ◦C until the extraction of DNA for genetic analyses. Physicochemical analyses and enzyme assays were conducted within five days after sampling. A subsample was dried in an oven at 70 ◦C and the amount of litter dry matter was determined, with the litter mass estimated in grams per square meter (Table1)[24].

2.2. Litter and Soil Nutrients Analyses In the laboratory, total C, N, and P were analyzed for both soil and litter samples. For all three sites, 1 g of fresh litter was placed in a 50 mL beaker and 10 mL of distilled water was added. The mix was stirred for 30 min at 100 rpm, allowed to stand for 5 min, and the pH reading was performed with a potentiometer (Accumet basic AB15, Thermo-Fisher Scientific, Waltham, MA, USA). The litter samples were oven-dried at 70 ◦C for 72 h, subsequently ground with a mill (Retsch MM400, Haan, Germany), and sieved through a 40 mesh. Similarly, soil samples were oven-dried and ground. Total N and P were determined following acid digestion in a mixture of concentrated H2SO4 and K2SO4 plus CuSO4, using the latter as a catalyst; N was determined by a micro-Kjeldahl method [40] and P by the molybdate colorimetric method following ascorbic acid reduction [41]. The extract was measured by colorimetry in an autoanalyzer (Bran-Luebbe; Nordestedt, Germany). Carbon was analysed in a total carbon analyzer (UIC 5012; Chicago, IL, USA) and determined by colorimetric detection [42].

2.3. Enzyme Activity Assays The activity of ligninolytic enzymes in litter samples was determined in triplicate within five days of collection in a Nanodrop 2000c spectrophotometer (Thermo Scientific Inc., Waltham, MA, USA). Enzyme extracts were prepared from 0.5 g aliquots from each litter sample that were incubated for 15 min at room temperature in 30 mL of modified universal extraction buffer (MUB) [43] with continuous stirring. All reaction mixtures were incubated at 30 ◦C for 120 min. Enzyme activities were expressed in units (U), defined as µmoles of product formed from substrate per hour (µmol h−1), per gram of soil (U g−1).

2.3.1. Laccase (Lac; EC 1.10.3.2) Lac determination was performed following the procedure of Nagai et al. [44], by measuring the oxidation of ABTS (2,20-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) to its cation radical. The reaction mixture included 300 µL enzyme extract, 100 µL of 10 mM ABTS (Sigma-Aldrich, St. Louis, USA), 200 µL of 0.2 M sodium acetate buffer (pH 5), and 400 µL sterile distilled water. Absorbance determinations were made at 420 nm. Forests 2018, 9, 11 5 of 18

2.3.2. Lignin Peroxidase (LiP; EC 1.11.1.14) The LiP assay was conducted using the method of Tien and Kirk [45], which is based on the principle that LiP uses H2O2 to catalyze the oxidation of veratryl alcohol to veratraldehyde. The reaction mixture included 300 µL of enzyme extract, 200 µL of 0.2 M sodium acetate buffer (pH 5), 200 µL of 10 mM veratryl alcohol (Sigma-Aldrich), 200 µL of sterile distilled water, and 100 µL of 0.4 mM H2O2 (Analytyka, Nuevo Leon, Mexico). Absorbance was measured at the absorption peak of veratraldehyde at 310 nm.

2.3.3. Manganese Peroxidase (MnP; EC 1.11.1.13) The activity of MnP was measured using a method based on the oxidation of 2,6-dimethoxyphenol, as described by Martinez et al. [46]. The reaction mixture contained 300 µL of enzyme extract, 200 µL of 0.2 M sodium acetate buffer (pH 5), 200 µL of sterile distilled water, 100 µL of 0.1 mM MnSO4 (Sigma-Aldrich), 100 µL of 10 mM 2,6-dimethoxyphenol (Sigma-Aldrich), and 100 µL of 0.4 mM H2O2 (Analytyka). Absorbance was measured at 469 nm.

2.4. Statistical Analyses of Enzyme Activity Statistica v.9.0 software (StatSoft, Palo Alto, CA, USA) was used to conduct repeated-measures analysis of variance (RMANOVA) with the site as the between-subjects factor and season and the interaction site-season as the within-subject factor to test for differences in enzyme activity. When RMANOVA indicated significant factor effects, a mean comparison was performed with Tukey’s multiple comparison test [47].

2.5. DNA Extraction, PCR Assays, Cloning, and Sequencing For molecular analyses, genomic DNA was extracted from 5 g of litter using the FastPrep System with the FastDNA spin for soil kit (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer’s instructions. The DNA obtained was purified with the DNA Clean and Concentrator-5 kit (Zymo Research, Irvine, CA, USA). The ITS region of the nuclear ribosomal unit was amplified using ITS1/ITS4 primers [48]; the total reaction mixture volume was 25 µL and it contained 50 ng DNA, 10 mM Tris-HCl (pH 8.5), 1.5 mM MgCl2, 0.5 mM of each deoxynucleoside triphosphate (dATP, dCTP, dGTP, and dTTP), 0.5 µM of each primer, 2% bovine serum albumin (BSA; Thermo Scientific), and 0.5 U Taq DNA recombinant polymerase (Invitrogen, Carlsbad, CA, USA). The PCR conditions used were 94 ◦C for 5 min, 35 1-min cycles at 94 ◦C (denaturation), 62 ◦C for 1 min (annealing), and 72 ◦C for 1 min (extension), followed by 10 min at 72 ◦C. The amplification products were visualized on 2% agarose gel stained with SYBR® Safe (Invitrogen, Carlsbad, CA, USA). The amplification products were then cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Plasmids were recovered by alkaline lysis [49]. Finally, the cloned products were sequenced at Elim Biopharmaceuticals, Inc. (Heyward, CA, USA) with primer M13F (50-GTAAAACGACGGCCAG-30).

2.6. Bioinformatics and Fungal Community Analyses Of the 1013 clone product sequences obtained, seven were identified as chimeras using UCHIME in de novo mode [50] and discarded, leaving a total 1006 sequences for analysis. Curated sequences were deposited in GenBank with accession numbers KT581643 to KT581949. DOTUR [51] was used to group the sequences obtained from the ITS region of rDNA into operational taxonomic units (OTUs) at 97% similarity. For each OTU, the longest sequence was selected and the closest hits were identified using the Blastn algorithm in GenBank (National Center for Biotechnology Information, NCBI, http://www.ncbi.nlm.nih.gov/). The following indices were also calculated with the same package: Shannon-Weaver (H’) and Simpson (1-D) [52] diversity indices, Chao1 richness estimator [53], and rarefaction analysis of each clone library. The library coverage values were calculated by [1-(n/N)], Forests 2018, 9, 11 6 of 18 where n is the number of OTUs representing a single clone (singleton) and N is the number of totalForests OTUs 2018, 9, representing 11 the clones in the library [54]. The Jaccard index was calculated using6 of the 18 software SONS [55] to examine differences in the fungal community between sites and sampling dates. βsoftware-Diversity SONS between [55] fungalto examine communities differences was in estimated the fungal with community the UniFrac between package sites [56] byand performing sampling Principaldates. β‐Diversity Coordinates between Analysis fungal (PCoA) communities and Jackknife was Cluster estimated Environment with the UniFrac analysis. package [56] by performing Principal Coordinates Analysis (PCoA) and Jackknife Cluster Environment analysis. 3. Results 3. Results 3.1. Nutrients and Vegetation in Studied Plots 3.1. Nutrients and Vegetation in Studied Plots Litter mass decreased by around 30% in the disturbed plots (Table1), but the C and N concentrationsLitter mass of decreased litter were by similar around among 30 % all in plots. the However,disturbed the plots litter (Table of the 1), WP but site the had C a and lower N Pconcentrations concentration, of and litter therefore were similar higher among C:P and all N:Pplots. ratios. However, The C the concentration litter of the WP in the site soil had was a lower lower P inconcentration, WP than in and the MDtherefore and HDhigher sites, C:P whereas and N:P soil ratios. N concentration The C concentration showed in the the oppositesoil was lower pattern. in TheWP Pthan concentration in the MD and was HD lower sites, at whereas the MD soil site N where, concentration therefore, showed higher the C:P opposite and N:P pattern. ratios The were P alsoconcentration found. was lower at the MD site where, therefore, higher C:P and N:P ratios were also found. TheThe presencepresence ofof plantplant speciesspecies characteristiccharacteristic ofof degradeddegraded sitessites includingincluding EysenhardtiaEysenhardtia polystachyapolystachya (Ortega)(Ortega) Sarg.,Sarg., LoeseliaLoeselia mexicanamexicana (Lam.)(Lam.) Brand,Brand, andand CrotonCroton sp.sp. waswas moremore conspicuousconspicuous inin thethe MDMD andand HDHD sites,sites, whereaswhereas allall plantplant speciesspecies foundfound inin thethe WPWP sitesite werewere characteristiccharacteristic ofof undisturbedundisturbed areasareas (Supplementary(Supplementary TableTable S1). 1).

3.2.3.2. EnzymeEnzyme ActivityActivity TheThe activityactivity ofof thethe threethree enzymesenzymes waswas significantlysignificantly affectedaffected byby seasonseason butbut notnot forestforest disturbancedisturbance (Table(Table2 ).2). At At the the three three sites, sites, Lac Lac activity activity was was five five times times higher higher in in the the rainy rainy than than in in the the dry dry season season (Figure(Figure1 1a),a), the the activity activity of of LiP LiP decreased decreased in in the the rainy rainy season season (Figure (Figure1 b),1b), and and MnP MnP behaved behaved in in a a similar similar wayway toto LiP,LiP, with with its its activity activity being being significantly significantly higher higher in in the the dry dry than than in in the the rainy rainy season season (Figure (Figure1 c).1c).

TableTable 2. 2.F F( p(p)) values values from from the the RMANOVAs RMANOVAs of of enzymatic enzymatic activityactivity inin QuercusQuercus deserticoladeserticola litter in three forest sites with increasingforest sites levels with of increasing disturbance levels in centralof disturbance Mexico. in central Mexico.

Between Subjects Within Subjects Enzyme Between Subjects Within Subjects Enzyme Site Sampling Season Interaction (Site:Season) Site Sampling Season Interaction (Site:Season) Laccase 0.28 (0.75) 106.42 (<0.0001) 0.12 (0.88) LaccaseManganese peroxidase 0.28 (0.75)1.65 (0.23) 106.4224.37 (<0.0001) (<0.0001) 2.42 0.12 (0.13) (0.88) ManganeseLignin peroxidase peroxidase 1.653.55 (0.23) (0.061) 24.3752.73 (<0.0001) (<0.0001) 1.76 2.42 (0.21) (0.13) Lignin peroxidase 3.55 (0.061) 52.73 (<0.0001) 1.76 (0.21) Bold letters denote significant (p ≤ 0.05) differences. Bold letters denote significant (p ≤ 0.05) differences.

(a)

Figure 1. Cont.

Forests 20182018,, 99,, 11 11 7 of 18

(b)

(c)

Figure 1. EnzymeEnzyme activities activities in in litter litter samples samples from from three three forest forest sites with increasing levels of disturbance in central Mexico by sampling date. Enzyme activities for laccase (a), lignin peroxidase (b), and manganese peroxidase (c) are shownshown byby sitesite andand season.season. Capital letters denote significantsignificant differences between sites; sites; lowercase lowercase letters letters denote denote significant significant differences differences between between sampling sampling dates dates (p (≤ p0.05).≤ 0.05).

3.3. Analysis of Fungal Communities A total of 1006 sequences of the fungal ITS region region were obtained over the the two two sampling sampling periods: periods: 502 during the dry season (175 from the WP site, 170 from the MD site, and 157 from the HD site; coverage valuesvalues ofof 0.811, 0.811, 0.823, 0.823, and and 0.892, 0.892, respectively), respectively), and and 504 504 during during the the rainy rainy season season (163 (163 from from the WPthe WP site, site, 173 from173 from the MD the site,MD andsite, 168and from 168 from the HD the site; HD coverage site; coverage values values of 0.816, of 0.816, 0.890, 0.890, and 0.887, and respectively).0.887, respectively). The coverage The coverage value ofvalue each of library each library above above 0.8 suggests 0.8 suggests that they that represented they represented the major the fungalmajor fungal phyla present phyla present in the litter in the samples. litter samples. In congruence In congruence with the coverage with the values coverage obtained, values rarefaction obtained, curvesrarefaction showed curves a tendency showed toa tendency slow their to increase slow their as theincrease sampling as the effort sampling increased effort (Figure increased2). (Figure 2).

Forests 2018, 9, 11 8 of 18 Forests 2018, 9, 11 8 of 18 Forests 2018, 9, 11 8 of 18

Figure 2. RarefactionRarefaction curves of a number number of fungal fungal OTUs OTUs found in in samples samples of QuercusQuercus deserticola litterlitter Figure 2. Rarefaction curves of a number of fungal OTUs found in samples of Quercus deserticola litter from forestforest sitessites with with increasing increasing levels levels of of disturbance disturbance (well (well preserved: preserved: WP; WP; moderately moderately disturbed: disturbed: MD; from forest sites with increasing levels of disturbance (well preserved: WP; moderately disturbed: MD;heavily heavily disturbed: disturbed: HD) HD) in central in central Mexico Mexico in the in dry the (D) dry season (D) season and rainy and rainy (W) season. (W) season. MD; heavily disturbed: HD) in central Mexico in the dry (D) season and rainy (W) season. When all samples were examined together, we observed a decrease in OTU richness with an When all samples were examined together, we observed a decrease in OTU richness with an increasing degree of disturbance: regardless regardless of of sampling sampling date date and and using using a a 97 97% % similarity threshold, threshold, increasing degree of disturbance: regardless of sampling date and using a 97 % similarity threshold, the WP and MD sites showed a similar richness with 95 and 93 OTUs, respectively, while 80 OTUs the WP and MD sites showed a similar richness with 95 and 93 OTUs, respectively, while 80 OTUs were identifiedidentified in the HD site (Table(Table3 ).3). WhenWhen sequencessequences werewere examinedexamined forfor eacheach samplingsampling datedate were identified in the HD site (Table 3). When sequences were examined for each sampling date separately, in in the the dry dry season, season, the the WP WP site site showed showed the the highest highest richness richness with with 58 OTUs, 58 OTUs, followed followed by the by separately, in the dry season, the WP site showed the highest richness with 58 OTUs, followed by the MDthe MDsite sitewith with 54 OTUs, 54 OTUs, and andthe HD the site HD with site with 43 OTUs. 43 OTUs. For the For rainy the rainy season season samples, samples, 56 OTUs 56 OTUs were MD site with 54 OTUs, and the HD site with 43 OTUs. For the rainy season samples, 56 OTUs were identifiedwere identified in the inWP the site, WP and site, 49 and 45 49 OTUs and 45 in OTUs the MD in and the MDHD sites, and HD respectively. sites, respectively. For both sampling For both identified in the WP site, and 49 and 45 OTUs in the MD and HD sites, respectively. For both sampling dates,sampling both dates, diversity both diversityindices (Shannon indices (Shannon-Weaver‐Weaver and Simpson) and Simpson) ranked the ranked diversity the diversity of sites ofin sites the dates, both diversity indices (Shannon-Weaver and Simpson) ranked the diversity of sites in the followingin the following order order(from (fromhighest highest to lowest): to lowest): WP, WP,MD, MD, and and HD HD (Table (Table 3).3). This This ranking ranking is is also inin following order (from highest to lowest): WP, MD, and HD (Table 3). This ranking is also in agreement with the number of singletons identified identified for each site. In In all three sites, the Chao1 index agreement with the number of singletons identified for each site. In all three sites, the Chao1 index showed thatthat the the observed observed richness richness was was lower lower than thethan estimated the estimated richness, richness, but that abut greater that proportiona greater showed that the observed richness was lower than the estimated richness, but that a greater proportionof the estimated of the species estimated had species been found had been in the found HD sitein the and HD the site MD and site the in MD the site rainy in season.the rainy season. proportion of the estimated species had been found in the HD site and the MD site in the rainy season. Table 3.3.Number Number of of fungal fungal OTUs, OTUs, singletons, singletons, and diversityand diversity indices indices for three for forest three sites forest with sites increasing with Table 3. Number of fungal OTUs, singletons, and diversity indices for three forest sites with increasinglevels of disturbance levels of disturbance in central Mexico in central in twoMexico contrasting in two contrasting seasons. seasons. increasing levels of disturbance in central Mexico in two contrasting seasons. Well Preserved (WP) Moderately Disturbed (MD) Heavily Disturbed (HD) Well Preserved (WP) Moderately Disturbed (MD) Heavily Disturbed (HD) Dry Rainy Dry Rainy Dry Rainy Dry Rainy Dry Rainy Dry Rainy No. of OTUs 58 56 54 49 43 45 No. of OTUs 58 56 54 49 43 45 No. of singletons 33 30 30 19 18 19 No. of singletons 33 30 30 19 18 19 Sequences per group Sequences per group Ascomycota 145 116 113 124 83 133 Ascomycota 145 116 113 124 83 133 Basidiomycota 17 29 21 37 25 28 Basidiomycota 17 29 21 37 25 28 Unidentified 16 19 39 12 49 7 Unidentified 16 19 39 12 49 7 Shannon‐Weaver (H’) 3.39 3.51 3.16 3.24 3.16 3.12 Shannon-Weaver (H’) 3.39 3.51 3.16 3.24 3.16 3.12 Simpson (1‐D) 0.96 0.93 0.94 0.93 0.90 0.92 Simpson (1-D) 0.96 0.93 0.94 0.93 0.90 0.92 Chao1 100 103 98 61 60 60 Chao1 100 103 98 61 60 60 Independently of the abundance, some taxa were shared between sites: the WP site shared 15 Independently of the abundance, some taxa were shared between sites: the WP site shared 15 taxa withIndependently the MD site of theand abundance, three taxa with some the taxa HD were site, shared whereas between the MD sites: site the shared WP site 20 shared taxa with 15 taxa the taxa with the MD site and three taxa with the HD site, whereas the MD site shared 20 taxa with the HDwith site. the MD Despite site and the three similar taxa number with the of HD OTUs site, whereas identified the in MD the site WP shared and 20 MD taxa sites, with there the HD were site. HD site. Despite the similar number of OTUs identified in the WP and MD sites, there were differencesDespite the in similar the order number of the of most OTUs abundant identified taxa in the at each WP andsite MDand sites,in the there abundance were differences of shared taxa. in the differences in the order of the most abundant taxa at each site and in the abundance of shared taxa. orderThe of themajority most abundantof OTUs belonged taxa at each to the site phylum and in the Ascomycota abundance (71 of shared%) followed taxa. by Basidiomycota The majority of OTUs belonged to the phylum Ascomycota (71 %) followed by Basidiomycota (16 %);The the majority remaining of OTUs 13 % were belonged allocated to the to phylumunidentified Ascomycota non‐cultivated (71%) followedfungi. This by pattern Basidiomycota of many (16 %); the remaining 13 % were allocated to unidentified non-cultivated fungi. This pattern of many more(16%); OTUs the remaining belonging 13% to Ascomycota were allocated than to to unidentified Basidiomycota non-cultivated was observed fungi. in the This three pattern sites of and many in more OTUs belonging to Ascomycota than to Basidiomycota was observed in the three sites and in both sampling dates (Table 3). In general, the most abundant taxa orders in the WP site were both sampling dates (Table 3). In general, the most abundant taxa orders in the WP site were Capnodiales (18 %), Hypocreales (17 %), and Pleosporales (16 %), whereas in the MD site they were Capnodiales (18 %), Hypocreales (17 %), and Pleosporales (16 %), whereas in the MD site they were

Forests 2018, 9, 11 9 of 18 moreForests 2018 OTUs, 9, 11 belonging to Ascomycota than to Basidiomycota was observed in the three sites9 andof 18 in both sampling dates (Table3). In general, the most abundant taxa orders in the WP site were CapnodialesPleosporales (18%),(19 %), Hypocreales Capnodiales (17%), (13 %), and and Pleosporales Agaricales (16%), (6 %), whereas and in in the the HD MD site site they were Pleosporales (19%),(25 %), Capnodiales Capnodiales (13%), (13 and%), Agaricalesand Thelephorales (6%), and (6 in the%). HDTaking site theysampling were Pleosporalesseason into (25%),account, Capnodiales at the WP site, (13%), Hypocreales and Thelephorales (30 %) and (6%). Capnodiales Taking sampling (27 %) were season the into most account, abundant at the orders WP site,in the Hypocreales dry season, while (30%) Pleosporales and Capnodiales (23 %) (27%) and Thelephorales were the most (10 abundant %) dominated orders in in the the rainy dry season,season while(Figure Pleosporales 3). In the MD (23%) site, and Capnodiales Thelephorales (25 (10%)%) and dominated Hypocreales in the (7 rainy%) dominated season (Figure in the3). dry In the season, MD site,and Pleosporales Capnodiales (25%)(34 %) and and Hypocreales Thelephorales (7%) (8 %) dominated were important in the dry in the season, rainy and season. Pleosporales Finally, in (34%) the andHD Thelephoralessite, Capnodiales (8%) (23 were %) important and Sordariales in the rainy (13 season. %) were Finally, dominant in the HDin the site, dry Capnodiales season, while (23%) andPleosporales Sordariales (40 (13%) %) and were Tubeufiales dominant (8 in %) the dominated dry season, in while the rainy Pleosporales season (Figure (40%) and 3). Tubeufiales (8%) dominatedThe Jaccard in the rainyindex season was used (Figure to3 ).evaluate the similarity between sites in terms of fungal communityThe Jaccard composition index was (Table used 4). to evaluateThe results the revealed similarity significant between sites changes in terms in the of composition fungal community of the compositionlitter fungal community (Table4). The of results a given revealed site between significant the dry changes and rainy in the seasons. composition The WP of thesite litter showed fungal the communityhighest between of a‐ givendates similarity site between (J = the0.253) dry and and the rainy other seasons. two sites The showed WP site even showed larger thedifferences highest between-datesbetween sampling similarity dates, (J with = 0.253) Jaccard and index the other values two of sites 0.102 showed and 0.099 even for larger the differences MD and HD between sites, samplingrespectively. dates, with Jaccard index values of 0.102 and 0.099 for the MD and HD sites, respectively.

Figure 3. Abundance ofof differentdifferent fungi fungi orders orders in in litter litter samples samples from from forest forest sites sites with with different different levels levels of disturbanceof disturbance (well (well preserved: preserved: WP; WP; moderately moderately disturbed: disturbed: MD; heavilyMD; heavily disturbed: disturbed: HD) in HD) central in Mexicocentral inMexico two contrasting in two contrasting seasons seasons (dry season: (dry D;season: rainy D; season: rainy W).season: Un. W). = unidentified. Un. = unidentified.

Table 4.4. JaccardJaccard similaritysimilarity indexindex of fungalfungal OTUs inin forestforest sitessites withwith differentdifferent levelslevels ofof disturbancedisturbance (well(well preserved:preserved: WP; moderately disturbed: MD; heavily disturbed: HD) in central Mexico in twotwo contrasting seasons (dry(dry season:season: D; rainy season: W). WP‐W MD‐D MD‐W HD‐D HD‐W WP-W MD-D MD-W HD-D HD-W WP‐D 0.253 0.352 0.099 0.296 0.104 WP-DWP‐ 0.253W 0.3520.160 0.301 0.099 0.133 0.296 0.304 0.104 WP-WMD‐D 0.160 0.102 0.301 0.308 0.133 0.096 0.304 MD-DMD‐W 0.102 0.106 0.308 0.667 0.096 MD-W 0.106 0.667 HD‐D 0.099 HD-D 0.099 WP, well preserved site; MD, moderately disturbed site; HD, heavily disturbed site; D, dry season; WP, well preserved site; MD, moderately disturbed site; HD, heavily disturbed site; D, dry season; and W, W, rainy rainy season. season. Although, in general, Basidiomycota were not among the most abundant taxa in the studied sites,Although, the comparison in general, of structural Basidiomycota changes were among not communities among the most is interesting abundant because taxa in members the studied of sites,this group the comparison are the main of structural producers changes of laccase among and communities peroxidases. is The interesting main orders because of membersBasidiomycetes of this found in the three study sites were Thelephorales and Agaricales, but their abundance was different between study sites and sampling seasons. The abundance of Thelephorales increased during the transition between the dry season to the wet season in WP and MD sites from 2 % to 35 % and from 9 % to 22 %, respectively; in the HD site, the abundance of these taxa decreased from 23 % in the dry

Forests 2018, 9, 11 10 of 18 group are the main producers of laccase and peroxidases. The main orders of Basidiomycetes found in the three study sites were Thelephorales and Agaricales, but their abundance was different between study sites and sampling seasons. The abundance of Thelephorales increased during the transition betweenForests 2018 the, 9, 11 dry season to the wet season in WP and MD sites from 2% to 35% and from 9% to10 22%, of 18 respectively; in the HD site, the abundance of these taxa decreased from 23% in the dry season to 17% inseason the wet to season.17 % in In the the wet case season. of Agaricales, In the WP case and of MD Agaricales, sites maintained WP and the MD abundance sites maintained of this order the betweenabundance sampling of this order dates (20%between and sampling 17%, respectively), dates (20 % but and the 17 abundance %, respectively), of Agaricales but the in abundance the HD site of increasedAgaricales from in the 4% HD in the site dry increased season from to 15% 4 % in in the the wet dry season. season The to 15 orders % in Polyporales, the wet season. Auriculariales, The orders andPolyporales, Atractiellales Auriculariales, were only found and inAtractiellales the WP site, were whereas only the found Tremellales in the and WP Sporidiobolales site, whereas were the onlyTremellales observed and in Sporidiobolales the MD site. The were HD siteonly did observed not present in the an MD exclusive site. The order, HD butsite there did not was present a greater an abundanceexclusive order, of unidentified but there was Basidiomycetes. a greater abundance The PCoA of unidentified of the sequences Basidiomycetes. obtained The showed PCoA a of clear the separationsequences obtained between theshowed dry anda clear rainy separation season samples between along the dry the and first rainy component, season samples which accounted along the forfirst 35% component, of the total which variance. accounted Component for 35 % 2 separatedof the total the variance. WP site Component from the MD 2 separated and HD sites the onWP both site samplingfrom the MD dates and (Figure HD sites4). on both sampling dates (Figure 4).

FigureFigure 4.4. Principal coordinates analysesanalyses showingshowing thethe relationships betweenbetween fungalfungal communitiescommunities fromfrom forestforest sitessites withwith increasingincreasing levelslevels ofof disturbancedisturbance (well(well preserved:preserved: WP;WP; moderatelymoderately disturbed:disturbed: MD; heavilyheavily disturbed:disturbed: HD)HD) inin centralcentral MexicoMexico inin twotwo contrastingcontrasting seasons seasons (dry (dry season: season: D; D; rainy rainy season:season: W).W).

4.4. DiscussionDiscussion InIn thisthis work, we compared compared the the ligninolytic ligninolytic enzyme enzyme activities activities and and the the overall overall fungal fungal richness richness of ofthe the litter litter community community from from three three forest forest fragments fragments dominated dominated by by Quercus deserticola withwith differentdifferent disturbancedisturbance levelslevels andand inin twotwo contrastingcontrasting seasons seasons (dry (dry and and rainy) rainy) in in central central Mexico. Mexico. TheThe hypothesishypothesis ofof thethe C:NC:N ratioratio beingbeing thethe majormajor driverdriver ofof litterlitter decompositiondecomposition inin forestforest ecosystemsecosystems [ [57]57] has has been been recently recently questioned questioned [58 [58].]. Litter Litter C:N C:N ratios ratios in in the the studied studied sites sites were were similar similar to thoseto those reported reported for thefor the litter litter from from other other oak species,oak species, including includingQ. ilex Q.L. ilex [58 L.], Q.[58], petraea Q. petraea(Matt.) (Matt.) Liebl. Liebl. [59], and[59],Q. and serrata Q. serrataThunb. Thunb. ex Murray ex Murray after after one year one ofyear decomposition of decomposition [60]. [60]. Most Most sites sites in this in studythis study had similarhad similar litter litter and soil and nutrient soil nutrient contents contents and ratios, and ratios, thus suggesting thus suggesting that decomposition that decomposition rates might rates bemight similar be similar between between them. However,them. However, the slightly the slightly greater greater N in the N soilin the of thesoil wellof the preserved well preserved site could site promotecould promote differences differences in the litter in the decomposition litter decomposition rate in relationrate in relation to the disturbed to the disturbed sites. It has sites. recently It has beenrecently proposed been proposed that the decomposition that the decomposition rate in N-poor rate in litter N‐poor will litter increase will whenincrease lying when over lying N-rich over soil, N‐ duerich tosoil, the due translocation to the translocation of N from of soil N tofrom litter soil [58 to]. litter [58]. EachEach sitesite hadhad plantplant speciesspecies characteristiccharacteristic ofof itsits disturbancedisturbance status,status, thusthus creatingcreating differentdifferent mixesmixes ofof litter,litter, which,which, duedue toto differencesdifferences inin NN availability, availability, labile/recalcitrant labile/recalcitrant CC sources,sources, andand secondarysecondary metabolitesmetabolites [ 61[61],], is ais determining a determining factor influencingfactor influencing the fungal the litter fungal community litter structure.community Comparative structure. Comparative decomposition analysis involving oak species shows contrasting results; whereas some studies document that Quercus spp. litter has slower decomposition rates than the litter of other plant species [62], and some authors found higher decomposition rates in oaks [63,64]. Quercus spp. litter recalcitrance has been attributed to high tannin and phenol concentrations, a poorer quality of long‐ lived leaves, and sclerophyllous leaf properties [62]. However, synergistic interactions in mixed litter could translocate nutrients from the labile to the recalcitrant substrates in the mix [61]. In this regard, the litter of Fraxinus uhdei (Wenz.) Lingelsh is considered to be of high quality due to its high nutrient

Forests 2018, 9, 11 11 of 18 decomposition analysis involving oak species shows contrasting results; whereas some studies document that Quercus spp. litter has slower decomposition rates than the litter of other plant species [62], and some authors found higher decomposition rates in oaks [63,64]. Quercus spp. litter recalcitrance has been attributed to high tannin and phenol concentrations, a poorer quality of long-lived leaves, and sclerophyllous leaf properties [62]. However, synergistic interactions in mixed litter could translocate nutrients from the labile to the recalcitrant substrates in the mix [61]. In this regard, the litter of Fraxinus uhdei (Wenz.) Lingelsh is considered to be of high quality due to its high nutrient concentrations and low concentrations of lignin and soluble polyphenols, and because it harbors a microbial community that produces fewer enzymes involved in N and P acquisition and more enzymes involved in cellulose degradation [65]. Thus, F. uhdei in the well preserved site should provide resources to cellulo-hydrolytic Ascomycota that provide labile nutrients to sustain the litter decomposition of recalcitrant components. It has also been documented that the litter of E. polystachya—a woody plant species found in both disturbed sites studied—has a C:N ratio of 15.1, but its decay rate is slow compared to that of other tree species, with decomposition rates thought to be associated with microenvironmental conditions under its canopy, as well as with its lignin and polyphenol content [66]. These conditions suggest that plant species associated with the well preserved site might increase litter decomposition rates and that plants associated with the disturbed sites might slow it down; a possibility that needs to be further evaluated. To the best of our knowledge, there are no studies documenting litter ligninolytic enzyme activities of fungal communities related to F. uhdei and E. polystachya—or of other plants species associated with Q. deserticola in the studied sites, which requires further study linking mycobiota, enzyme activities, and nutrient cycles in litter. Laccase activity in Q. deserticola litter samples was higher during the rainy than during the dry season, while peroxidase (LiP and MnP) activities showed the opposite pattern. In Q. petraea litter, Lac showed low activity in spring (May) and high activity levels in summer (July), while MnP exhibited a higher activity in spring than in summer [67]. A similar trend in Lac activity was observed for Q. ilex litter, but not for MnP [68]; however, for this same oak species, Kellner et al. [69] reported the higher MnP activity in litter subject to drought relative to control samples. Thus, our results are in agreement with previous findings of seasonal activity patterns of ligninolytic enzyme activities in oak forests. Changes in the activity of lignocellulolytic enzymes in forest soil and litter are correlated with environmental factors, including temperature, moisture, and pH [70]. However, Criquet et al. [68] found no correlation between the activity of Lac and MnP and abiotic factors such as temperature, humidity, and pH in Q. ilex litter. Thus, besides physical and climatic variables, chemical changes in litter composition [68], variations in fungal biomass [71], and changes in fungal community composition [72] can explain the changes in enzyme activity patterns. The lack of differences in enzyme activities between the sites studied could be attributed to functional redundancy in fungal communities: different species being capable of producing ligninolytic enzymes in different ecological contexts [73]. Recently, soil microbial community redundancy—also called functional convergence—has been described between under-canopy and open areas of Q. ilex forest fragments harboring different bacterial and fungal communities, but showing similar metabolic patterns [32]. Thus, it is quite possible that the functional redundancy of basidiomycetes could explain the similar ligninolytic activities found in the Q. deserticola litter along a disturbance gradient (see below). The fungal community we described is highly consistent with previously studied fungal litter communities at different taxonomic levels. In our study, 70% of the sequences were associated with the phylum Ascomycota, 15% were Basidiomycota, and 15% remained as unidentified fungi. A small percentage (0.2%) of the sequences showed a relation with the former phylum Zygomycota, now invalid; however, because it was not possible to know if these sequences were within Glomeromycota or Mucoromycotina, we decided to group them with unidentified fungi. In a previous analysis conducted by the authors, we found the same proportions of Ascomycota and Basidiomycota [24], supporting the representativeness of our present data. These abundances agree with the relative percentages reported in previous investigations of mixed hardwood litter in which the Forests 2018, 9, 11 12 of 18

Ascomycota accounted for 62–85%, the Basidiomycota for 15–36%, and the Glomeromycota for up to 2% of the total fungal community [74]. In Q. petraea litter, the composition was different, with Ascomycota representing 42% and Basidiomycota 48% of the fungal community [67], the lowest abundances corresponding to Glomeromycota and Mucoromycotina. Gibberella, Teratosphaeria, and Cladosporium were among the most abundant genera during the dry season. Such results are consistent with previous findings for fungi colonizing Quercus spp. leaves and litter. Cladosporium spp. have been described as phyllosphere fungi in the early stages of litter decomposition in Q. leucotrichophora A. Camus [75], Q. rotundifolia Lam. [76], and Q. petraea [72] forests. Teratosphaeria species have been found in the phyllosphere of Q. petraea [72] and anamorphs of Gibberella (Fusarium) species have been isolated from senescent and early decomposition stages of Q. rotundifolia litter [76]; however, Fusarium has been found as a later colonizer of Q. myrsinaefolia Blume litter [77]. Phoma and Pyrenochaeta were the most abundant groups in the rainy season, with the former noted as phyllosphere fungi in Quercus spp. [72,77] and occurring in the early stages of litter decomposition [75,77]. All these genera within Ascomycota contribute as producers of hydrolytic enzymes acting on plant cell wall polysaccharides [78,79]. The fungal taxa associated with the degradation of lignin in litter are within Basidiomycota. Among the taxa of Basidiomycota we found in the studied sites, members of the family Thelephoraceae—particularly Tomentella—were the most abundant OTUs in rainy-season samples. Our previous analysis of litter of Q. deserticola found Tomentella sp. to be a prevalent taxon [24], which shows a wide distribution in Q. deserticola forests. Interestingly, it has been documented that Tomentella sublilacina forming ectomycorrhizal (ECM) associations with Q. robur L. increased the hydrolytic and laccase enzyme activities in a thinned tree stand, but not so in a disturbed stand of the same tree species [80]; this shows both that appropriate management will conserve fungal diversity and function, and that some of the fungal-plant biotrophic interactions might be used as indicators of forest performance. Regarding the orders of Basidiomycota sampled here, genomic phylogenetic analysis has shown that white rot Auriculariales and Polyporales possess high numbers of MnP and LiP genes, Agaricales retained low gene numbers, and Tremellales, Atractiellales, and Sporidiobolales have lost the genes coding such enzymes [81]. Thus, our present results show that the litter of well preserved Q. deserticola forest sustains the highest diversity of well-identified ligninolytic fungi, mainly of the strongest lignin degraders. In the previous study conducted in Q. deserticola litter [24], Corticiales and Thelephorales were the only orders of Basidiomycota found, indicating the representativeness of the samples herein analyzed. Despite the differences we observed in the structure of the ligninolytic fungal community, we found no differences in the enzyme activities of Lac and peroxidases among the three studied sites. It must be taken into account that both Agaricales and Thelephorales were found along the forest degradation gradient we evaluated, and that unidentified basidiomycetes were only found in the disturbed site. Although many members of the two former taxa are considered ECM, they may be contributing, along with the unidentified taxa, to sustain ligninolytic activities in the litter of disturbed sites. Recent genomic evidence shows that certain ECM taxa have retained genes for Lac and MnP enzymes from their saprotrophic ancestors [82,83]. However, it has been postulated that ECM fungi are not facultative saprotrophs using lignin as the principal source of metabolic C, but use the conservation of Lac and MnP activities for mobilizing N locked up in non-hydrolysable, recalcitrant organic matter complexes [84]. Despite this, the ligninolytic activity of ECM fungi might play a central role in the turnover and stabilization of organic matter, influencing the C and N dynamics of temperate forest ecosystems [83,84]. Thus, it is possible that ECM fungi replaced saprobic guilds in the perturbed forest sites, something that deserves detailed assessment in future studies. We found that the fungal community of Quercus deserticola litter was influenced both by the sampling date and by the degree of forest disturbance. Seasonal changes of whole fungal soil and litter communities have been documented in Quercus spp. forests [67,74]. The composition of the fungal soil community in pine-oak and oak–hickory stands was found to be closely associated with changes Forests 2018, 9, 11 13 of 18 in soil nutrient status and specific changes in edaphic properties might explain the observed shifts in the microbial community [85]. Thus, previously noted seasonal changes in N content and the C:N ratio of the Q. deserticola litter partially explain seasonal changes in the fungal community structure. As stated above, the highest richness of fungal OTUs was found in the well preserved site, suggesting that conservation status affects the diversity of the fungal community in Q. deserticola litter. Historically, pine-oak forest fragmentation in the state of Michoacán has been associated with agricultural expansion, grazing, and logging for wood and charcoal extraction [34–36], as is the case of the studied area. It has been previously documented that Quercus spp. forests management practices [62] and fragmentation cause changes in soil and litter fungal community structure [28]. On one side, in a Q. ilex forest, Richard et al. [86] documented a significant correlation between the species richness of macroscopic saprobic and ECM fungi and tree density. On the other side, Azul et al. [27,87] found that logging, soil tillage, and permanent grazing reduced the macroscopic (mainly ECM) fungal community in Q. suber ecosystems. In the context of the socioeconomic needs of rural communities in developing countries like Mexico, a balance must be found between practices of forest use and conservation so that biodiversity is conserved while the energy and food requirements of the population are fulfilled [88]. In the state of Michoacán, the compromise between the use and conservation of forests has been analyzed in indigenous Purépecha artisanal and peasant rural communities [35]. However, forest management promoting conservation has largely ignored litter and fungi as vital components of forest functioning. Appropriate stand management of Quercus spp. forests—such as logging without soil tillage and grazing—has been documented to preserve fungal diversity [27,87]. Furthermore, within the studied area, models for sustainable charcoal extraction have been developed [36]. Additionally, our present results and those from previous work show that some of the orders and genera of fungi present in the Quercus spp. litter remain constant despite geographical distance and differences in climatic conditions. The data increases our understanding of the geographical range of fungi associated with Quercus spp. litter, and will enable the generation of successional models and increase our understanding of fungal community responses to management, restoration, and climatic change [89]. Further studies are needed to assess the interactions between environment and land use variables affecting the Q. deserticola litter fungal community. Knowledge derived from such studies will prove useful for designing better management practices so that the socioeconomic demands of the rural population are satisfied, in addition to preserving the functions and biodiversity of the forest ecosystem [31].

5. Conclusions The fungal community structure and the decomposition process of Q. deserticola litter in Mexican forests are highly dynamic. The activity of three major ligninolytic enzymes showed similarities among them. Therefore, in the near future, it might be possible to formulate a model of fungal succession and enzyme dynamics in oak forest litter, as has been achieved by similar studies in different geographic areas. Such models can guide the formulation of appropriate management and conservation strategies for oak forests. In particular, further studies are needed to better understand the interactions taking place in Q. deserticola forests between forest management, litter chemistry, seasonal changes, the composition of fungal communities, and their enzyme activities.

Supplementary Materials: The following are available online at www.mdpi.com/1999-4907/9/1/11/s1, Table S1: Vegetation found in the study plots used as indicator of conservation or disturbance. Acknowledgments: This project was supported by grants awarded by the PAPIIT program, Universidad Nacional Autónoma de México (UNAM; IN213113 and IV201015) and Consejo Nacional de Ciencia y Tecnología (CONACYT) 240136 to K. Oyama. We acknowledge Rodrigo Velázquez-Durán for chemical analyses. We are grateful to three anonymous reviewers and the Guest editor, Dr. Francis Brearley, that contributed to substantially improving the manuscript. Author Contributions: K.O. and G.V.-M. conceived and designed the study and the experiments; J.A.R.-C. determined enzyme activities, isolated litter DNA, and performed PCR assays and fungal community analysis; Forests 2018, 9, 11 14 of 18

R.A.-R. aided in selection and characterization of studied plots, and identified and described plant species in the plots; F.G.-O. performed litter nutrients determination and analyzed the data; M.S.V.-G. contributed reagents, materials, analysis tools, and ITS library construction; K.O., G.V.-M., and F.G.-O. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Swift, M.J.; Heal, O.W.; Anderson, J.M. Decomposition in Terrestrial Ecosystems; Blackwell: London, UK, 1979; p. 372. 2. Hättenschwiler, S.; Tiunov, A.V.; Scheu, S. Biodiversity and litter decomposition in terrestrial ecosystems. An. Rev. Ecol. Evol. Syst. 2005, 36, 191–218. [CrossRef] 3. Peay, K.G.; Kennedy, P.G.; Talbot, J.M. Dimensions of biodiversity in the Earth mycobiome. Nat. Rev. Microbiol. 2016, 14, 434–447. [CrossRef] [PubMed] 4. Zak, D.R.; Pregitzer, K.S.; Burton, A.J.; Edwards, I.P.; Kellner, H. Microbial responses to a changing environment: Implications for the future functioning of terrestrial ecosystems. Fungal Ecol. 2011, 4, 386–395. [CrossRef] 5. Osono, T. Diversity and functioning of fungi associated with leaf litter decomposition in Asian forests of different climatic regions. Fungal Ecol. 2011, 4, 375–385. [CrossRef] 6. Peršoh, D. Plant-associated fungal communities in the light of meta’omics. Fungal Divers. 2015, 75, 1–25. [CrossRef] 7. Urbanová, M.; Šnajdr, J.; Baldrian, P. Composition of fungal and bacterial communities in forest litter and soil is largely determined by dominant trees. Soil Biol. Biochem. 2015, 84, 53–64. [CrossRef] 8. Talbot, J.M.; Bruns, T.D.; Taylor, J.W.; Smith, D.P.; Branco, S.; Glassman, S.I.; Erlandson, S.; Vilgalys, R.; Liao, H.-L.; Smith, M.E.; et al. Endemism and functional convergence across the North American soil mycobiome. Proc. Natl. Acad. Sci. USA 2014, 111, 6341–6346. [CrossRef][PubMed] 9. Nixon, K.C. Infrageneric classification of Quercus (Fagaceae) and typification of sectional names. Ann. Sci. For. 1993, 50, 25–34. [CrossRef] 10. Kremer, A.; Casasoli, M.; Barreneche, T.; Bodénès, C.; Sisco, P.; Kubisiak, T.; Scalfi, M.; Leonardi, S.; Bakker, S.; Buiteveld, J.; et al. Fagaceae Trees. In Forest Trees, 1st ed.; Kole, C., Ed.; Springer: Berlin/Heidelberg, Germany, 2007; Volume 7, pp. 161–187. 11. Nixon, K.C. Global and Neotropical distribution and diversity of Oak (genus Quercus) and Oak forests. In Ecology and Conservation of Neotropical Montane Oak Forests; Kappelle, M., Ed.; Springer: Berlin/Heidelberg, Germany, 2006; pp. 3–13. 12. Aldrich, P.R.; Cavender-Bares, J. Quercus. In Wild Crop Relatives: Genomic and Breeding Resources; Kole, C., Ed.; Springer: Berlin/Heidelberg, Germany, 2011; pp. 89–129. 13. Rzedowski, J. Vegetación de México, 1st ed.; Limusa: Distrito Federal, México, 1978; 504p. 14. Gómez-Luna, B.E.; Rivera-Mosqueda, M.C.; Dendooven, L.; Vázquez-Marrufo, G.; Olalde-Portugal, V. Charcoal production at kiln sites affects C and N dynamics and associated soil microorganisms in Quercus spp temperate forests of central Mexico. Appl. Soil Ecol. 2009, 41, 50–58. [CrossRef] 15. Aguilar, R.; Guilardi, A.; Vega, E.; Skutsch, M.; Oyama, K. Sprouting productivity and allometric relationships of two oak species managed for traditional charcoal making in central Mexico. Biomass Bioenergy 2012, 36, 192–207. [CrossRef] 16. Chapa-Vargas, L.; Monzalvo-Santos, K. Natural protected areas of San Luis Potosí, Mexico: Ecological representativeness, risks, and conservation implications across scales. Int. J. Geogr. Inf. Sci. 2012, 26, 1625–1641. [CrossRef] 17. Asbjornsen, H.; Ashton, M.S.; Vogt, D.J.; Palacios, S. Effects of habitat fragmentation on the buffering capacity of edge environments in a seasonally dry tropical oak forest ecosystem in Oaxaca, Mexico. Agric. Ecosyst. Environ. 2004, 103, 481–495. [CrossRef] 18. Galicia, L.; Zarco-Arista, A.E.; Mendoza-Robles, K.I.; Palacio-Prieto, J.L.; García-Romero, A. Land use/cover, landforms and fragmentation patterns in a tropical dry forest in the southern Pacific region of Mexico. Singapore J. Trop. Geogr. 2008, 29, 137–154. [CrossRef] Forests 2018, 9, 11 15 of 18

19. García-Oliva, F.; Covaleda, S.; Gallardo, J.F.; Prat, C.; Velázquez-Durán, R.; Etchevers, J.D. Firewood extraction affects carbon pools and nutrients in remnant fragments of temperate forest at the Mexican Transvolcanic Belt. Bosque 2014, 35, 311–324. [CrossRef] 20. Rodríguez-Trejo, D.A.; Myers, R.L. Using oak characteristics to guide fire regime restoration in Mexican pine-oak and oak forests. Ecol. Restor. 2010, 28, 304–323. [CrossRef] 21. Bugalho, M.N.; Caldeira, M.C.; Pereira, J.S.; Aronson, J.; Pausas, J.G. Mediterranean cork oak savannas require human use to sustain biodiversity and ecosystem services. Front. Ecol. Environ. 2011, 9, 278–286. [CrossRef] 22. Oliver, T.H.; Morecroft, M.D. Interactions between climate change and land use change on biodiversity: Attribution problems, risks, and opportunities. WIREs Clim. Chang. 2014, 5, 317–335. [CrossRef] 23. Morris, S.J.; Friese, C.F.; Allen, M.F. Disturbance in natural ecosystems: Scaling from fungal diversity to ecosystem functioning. In Environmental and Microbial Relationships; Mycota, I.V., Druzhinina, I.S., Kubicek, C.P., Eds.; Springer: Berlin/Heidelberg, Germany, 2016; pp. 79–98. 24. Chávez-Vergara, B.; Rosales-Castillo, A.; Merino, A.; Vázquez-Marrufo, G.; Oyama, K.; García-Oliva, F. Quercus species control nutrients dynamics by determining the composition and activity of the forest floor fungal community. Soil Biol. Biochem. 2016, 98, 186–195. [CrossRef] 25. Morris, M.H.; Pérez-Pérez, M.A.; Smith, M.E.; Bledsoe, C.S. Multiple species of ectomycorrhizal fungi are frequently detected on individual oak root tips in a tropical cloud forest. Mycorrhiza 2008, 18, 375–383. [CrossRef][PubMed] 26. Van der Wal, A.; Geydan, T.D.; Kuyper, T.W.; de Boer, W. A thready affair: Linking fungal diversity and community dynamics to terrestrial decomposition processes. FEMS Microbiol. Rev. 2013, 37, 477–494. [CrossRef][PubMed] 27. Azul, A.M.; Sousa, J.P.; Agerer, R.; Martín, M.P.; Freitas, H. Land use practices and ectomycorrhizal fungal communities from oak woodlands dominated by Quercus suber L. considering drought scenarios. Mycorrhiza 2010, 20, 73–88. [CrossRef][PubMed] 28. Williams, R.J.; Hallgren, S.W.; Wilson, G.W.T. Frequency of prescribed burning in an upland oak forest determines soil and litter properties and alters the soil microbial community. For. Ecol. Manag. 2012, 265, 241–247. [CrossRef] 29. Trivedi, P.; Delgado-Baquerizo, M.; Trivedi, C.; Hu, H.; Anderson, I.C.; Jeffries, T.C.; Zhou, J.; Singh, B.K. Microbial regulation of the soil carbon cycle: Evidence from gene–enzyme relationships. ISME J. 2016, 10, 2593–2604. [CrossRef][PubMed] 30. Avis, P.G.; Gaswick, W.C.; Tonkovich, G.S.; Leacock, P.R. Monitoring fungi in ecological restorations of coastal Indiana, USA. Restor. Ecol. 2017, 25, 92–100. [CrossRef] 31. Purahong, W.; Kapturska, D.; Pecyna, M.J.; Schloter, M.; Buscot, F.; Hofrichter, M.; Krüger, D. Influence of different forest system management practices on leaf litter decomposition rates, nutrient dynamics and the activity of ligninolytic enzymes: a case study from Central European forests. PLoS ONE 2014, 9, e93700. [CrossRef][PubMed] 32. Flores-Rentería, D.; Rincón, A.; Valladares, F.; Yuste, J.C. Agricultural matrix affects differently the alpha and beta structural and functional diversity of soil microbial communities in a fragmented Mediterranean holm oak forest. Soil Biol. Biochem. 2016, 92, 79–90. [CrossRef] 33. Cornejo-Tenorio, G.; Sánchez-García, E.; Flores-Tolentino, M.; Santana-Michel, F.; Ibarra-Manríquez, G. Flora y vegetación del cerro El Águila, Michoacán, México. Bot. Sci. 2013, 91, 155–180. 34. Klooster, D. Forest transitions in Mexico: Institutions and forests in a globalized countryside. Prof. Geogr. 2003, 55, 227–237. [CrossRef] 35. Works, M.A.; Hadley, K.S. The cultural context of forest degradation in adjacent Purépechan communities, Michoacán, Mexico. Geogr. J. 2004, 170, 22–38. [CrossRef] 36. Castillo-Santiago, M.Á.; Ghilardi, A.; Oyama, K.; Hernández-Stefanoni, J.L.; Torres, I.; Flamenco-Sandoval, A.; Fernández, A.; Mas, J.F. Estimating the spatial distribution of woody biomass suitable for charcoal making from remote sensing and geostatistics in central Mexico. Energy Sust. Dev. 2013, 17, 177–188. [CrossRef] 37. López, E.; Bocco, G.; Mendoza, M.; Velázquez, A.; Aguirre-Rivera, J.R. Peasant emigration and land-use change at the watershed level: A GIS-based approach in central Mexico. Agric. Syst. 2006, 90, 62–78. [CrossRef] Forests 2018, 9, 11 16 of 18

38. Gentry, A.H. Diversity and floristic composition of Neotropical dry forests. In Seasonally Dry Tropical Forests; Bullock, S.H., Mooney, H.A., Medina, E., Eds.; Cambridge University Press: Cambridge, CA, USA, 1995; pp. 146–194. 39. Chávez-Vergara, B.; Merino, A.; Vázquez-Marrufo, G.; García-Oliva, F. Organic matter dynamics and microbial activity during decomposition of forest floor under two native Neotropical oak species in a temperate deciduous forest in Mexico. Geoderma 2014, 235, 133–145. [CrossRef] 40. Bremmer, J.M. Nitrogen-Total. In Methods of Soil Analyses Part 3: Chemical Analyses; Spark, D.L., Page, A.L., Summer, M.E., Tabatabai, M.A., Helmke, P.A., Eds.; Soil Science Society of America: Madison, WI, USA, 1996; pp. 1085–1121. 41. Murphy, J.; Riley, J.P. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta 1962, 27, 31–36. [CrossRef] 42. Huffman, E.W.D. Performance of a new automatic carbon dioxide coulometer. Microchem. J. 1977, 22, 567–573. [CrossRef] 43. Parham, J.A.; Deng, S.P. Detection, quantification and characterization of β-glucosaminidase activity in soil. Soil Biol. Biochem. 2000, 32, 1183–1190. [CrossRef] 44. Nagai, M.; Sato, T.; Watanabe, H.; Saito, K.; Kawata, M.; Enei, H. Purification and characterization of an extracellular laccase from the edible mushroom Lentinula edodes and decolorization of chemically different dyes. Appl. Microbiol. Biotechnol. 2002, 60, 327–335. [CrossRef][PubMed] 45. Tien, M.; Kirk, T.K. Lignin peroxidase of Phanerochaete chrysosporium. Methods Enzymol. 1988, 161, 238–249. [CrossRef] 46. Martinez, M.J.; Ruiz-Duenas, F.J.; Cuillen, F.; Martinez, A.T. Purification and catalytic properties of two manganese peroxidase isoenzymes from Pleurotus eryngii. Eur. J. Biochem. 1996, 237, 424–432. [CrossRef] [PubMed] 47. Von Ende, C.N. Repeated measures analysis: growth and other time-dependent measures. In Design and Analysis of Ecological Experiments; Scheiner, S.M., Gurevitch, J., Eds.; Chapman & Hall: New York, NY, USA, 1993; pp. 113–137. 48. White, T.J.; Bruns, T.; Lee, S.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols; Innins, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J., Eds.; Academic Press: Oxford, MA, USA, 1990; pp. 315–322. 49. Sambrook, J.; Russell, D.W. Molecular cloning. In A Laboratory Manual, 3rd ed.; Cold Spring Harbor Laboratory Press: New York, NY, USA, 2001; p. 2100. 50. Edgar, R.C.; Haas, B.J.; Clemente, J.C.; Quince, C.; Knight, R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 2011, 27, 2194–2200. [CrossRef][PubMed] 51. Schloss, P.D.; Handelsman, J. Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl. Environ. Microbiol. 2005, 71, 1501–1506. [CrossRef][PubMed] 52. Magurran, A.E. Ecological Diversity and Its Measurement; Chapman and Hall: London, UK, 1996; p. 192. 53. Chao, A. Estimating the population size for capture-recapture data with unequal catchability. Biometrics 1987, 43, 783–791. [CrossRef][PubMed] 54. Hu, L.; Cao, L.; Zhang, R. Bacterial and fungal taxon changes in soil microbial community composition induced by short-term biochar amendment in red oxidized loam soil. World J. Microbiol. Biotechol. 2014, 30, 1085–1092. [CrossRef][PubMed] 55. Schloss, P.D.; Handelsman, J. Introducing SONS, a tool for OTU-based comparisons of membership and structure between microbial communities. Appl. Environ. Microbiol. 2006, 72, 6773–6779. [CrossRef] [PubMed] 56. Lozupone, C.; Hamady, M.; Knight, R. UniFrac an online tool for comparing microbial community diversity in a phylogenetic context. BMC Bioinformatics 2006, 7, 371. [CrossRef][PubMed] 57. Hättenschwiler, S.; Coq, S.; Barantal, S.; Handa, I.T. Leaf traits and decomposition in tropical rainforests: revisiting some commonly held views and towards a new hypothesis. New Phytol. 2011, 189, 950–965. [CrossRef][PubMed] 58. Bonanomi, G.; Cesarano, G.; Gaglione, S.A.; Ippolito, F.; Sarker, T.; Rao, M.A. Soil fertility promotes decomposition rate of nutrient poor, but not nutrient rich litter through nitrogen transfer. Plant Soil 2017, 412, 397–411. [CrossRef] 59. Kubartová, A.; Ranger, J.; Berthelin, J.; Beguiristain, T. Diversity and decomposing ability of saprophytic fungi from temperate forest litter. Microb. Ecol. 2009, 58, 98–107. [CrossRef][PubMed] Forests 2018, 9, 11 17 of 18

60. Salamanca, E.F.; Kaneko, N.; Katagiri, S.; Nagayama, Y. Nutrient dynamics and lignocellulose degradation in decomposing Quercus serrata leaf litter. Ecol. Res. 1998, 13, 199–210. [CrossRef] 61. Gessner, M.O.; Swan, C.M.; Dang, C.K.; McKie, B.G.; Bardgett, R.D.; Wall, D.H.; Hättenschwiler, S. Diversity meets decomposition. Trends Ecol. Evol. 2010, 25, 372–380. [CrossRef][PubMed] 62. Sheffer, E.; Canham, C.D.; Kigel, J.; Perevolotsky, A. Countervailing effects on pine and oak leaf litter decomposition in human-altered Mediterranean ecosystems. Oecologia 2015, 177, 1039–1051. [CrossRef] [PubMed] 63. Arslan, H.; Guleryuz, G.; Kırmızı, S. Nitrogen mineralisation in the soil of indigenous oak and pine plantation forests in a Mediterranean environment. Eur. J. Soil Biol. 2010, 46, 11–17. [CrossRef] 64. Barba, J.; Lloret, F.; Yuste, J.C. Effects of drought-induced forest die-off on litter decomposition. Plant Soil 2016, 402, 91–101. [CrossRef] 65. Rothstein, D.E.; Vitousek, P.M.; Simmons, B.L. An exotic tree alters decomposition and nutrient cycling in a Hawaiian montane forest. Ecosystems 2004, 7, 805–814. [CrossRef] 66. Ceccon, E.; Sánchez, I.; Powers, J.S. Biological potential of four indigenous tree species from seasonally dry tropical forest for soil restoration. Agrofor. Syst. 2015, 9, 455–467. [CrossRef] 67. Voˇríšková, J.; Brabcová, V.; Cajthaml, T.; Baldrian, P. Seasonal dynamics of fungal communities in a temperate oak forest soil. New Phytol. 2014, 201, 269–278. [CrossRef][PubMed] 68. Criquet, S.; Farnet, A.M.; Tagger, S.; Le Petit, J. Annual variations of phenoloxidase activities in an evergreen oak litter: Influence of certain biotic and abiotic factors. Soil Biol. Biochem. 2000, 32, 1505–1513. [CrossRef] 69. Kellner, H.; Luis, P.; Pecyna, M.J.; Barbi, F.; Kapturska, D.; Krüger, D.; Zak, D.R.; Marmeisse, R.; Vandenbol, M.; Hofrichter, M. Widespread occurrence of expressed fungal secretory peroxidases in forest soils. PLoS ONE 2014, 9, e95557. [CrossRef][PubMed] 70. Baldrian, P.; Šnajdr, J.; Merhautová, V.; Dobiášová, P.; Cajthaml, T.; Valášková, V.Responses of the extracellular enzyme activities in hardwood forest to soil temperature and seasonality and the potential effects of climate change. Soil Biol. Biochem. 2013, 56, 60–68. [CrossRef] 71. Papa, S.; Pellegrino, A.; Fioretto, A. Microbial activity and quality changes during decomposition of Quercus ilex leaf litter in three Mediterranean woods. Appl. Soil Ecol. 2008, 40, 401–410. [CrossRef] 72. Voˇríšková, J.; Baldrian, P. Fungal community on decomposing leaf litter undergoes rapid successional changes. ISME J. 2013, 7, 477–486. [CrossRef][PubMed] 73. Schimel, J.P.; Schaeffer, S.M. Microbial control over carbon cycling in soil. Front. Microbiol. 2012, 3, 348. [CrossRef][PubMed] 74. O’Brien, H.E.; Parrent, J.L.; Jackson, J.A.; Moncalvo, J.M.; Vilgalys, R. Fungal community analysis by large-scale sequencing of environmental samples. Appl. Environ. Microbiol. 2005, 71, 5544–5550. [CrossRef] [PubMed] 75. Singh, S.P.; Pande, K.; Upadhyay, V.P.; Singh, J.S. Fungal communities associated with the decomposition of a common leaf litter (Quercus leucotrichophora A Camus) along an elevational transect in the Central Himalaya. Biol. Fert. Soils 1990, 9, 245–251. [CrossRef] 76. Sadaka, N.; Ponge, J.F. Fungal colonization of phyllosphere and litter of Quercus rotundifolia Lam in a holm oak forest (High Atlas, Morocco). Biol. Fertil. Soils 2003, 39, 30–36. [CrossRef] 77. Shirouzu, T.; Hirose, D.; Fukasawa, Y.; Tokumasu, S. Fungal succession associated with the decay of leaves of an evergreen oak, Quercus myrsinaefolia. Fungal Divers. 2009, 34, 87–109. 78. Baldrian, P.; Voˇríšková, J.; Dobiášová, P.; Merhautová, V.; Lisá, L.; Valášková, V. Production of extracellular enzymes and degradation of biopolymers by saprotrophic microfungi from the upper layers of forest soil. Plant Soil 2011, 338, 111–125. [CrossRef] 79. Schneider, T.; Keiblinger, K.M.; Schmid, E.; Sterflinger-Gleixner, K.; Ellersdorfer, G.; Roschitzki, B.; Richter, A.; Eberl, L.; Zechmeister-Boltenstern, S.; Riedel, K. Who is who in litter decomposition? Metaproteomics reveals major microbial players and their biogeochemical functions. ISME J. 2012, 6, 1749–1762. [CrossRef] [PubMed] 80. Mosca, E.; Montecchio, L.; Scattolin, L.; Garbaye, J. Enzymatic activities of three ectomycorrhizal types of Quercus robur L in relation to tree decline and thinning. Soil Biol. Biochem. 2007, 39, 2897–2904. [CrossRef] 81. Floudas, D.; Binder, M.; Riley, R.; Barry, K.; Blanchette, R.A.; Henrissat, B.; Martínez, A.T.; Otillar, R.; Spatafora, J.W.; Yadav, J.S.; et al. The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science 2012, 336, 1715–1719. [CrossRef][PubMed] Forests 2018, 9, 11 18 of 18

82. Bödeker, I.; Clemmensen, K.E.; Boer, W.; Martin, F.; Olson, Å.; Lindahl, B.D. Ectomycorrhizal Cortinarius species participate in enzymatic oxidation of humus in northern forest ecosystems. New Phytol. 2014, 203, 245–256. [CrossRef][PubMed] 83. Shah, F.; Nicolás, C.; Bentzer, J.; Ellström, M.; Smits, M.; Rineau, F.; Canbäck, B.; Floudas, D.; Carleer, R.; Lackner, G.; et al. Ectomycorrhizal fungi decompose soil organic matter using oxidative mechanisms adapted from saprotrophic ancestors. New Phytol. 2016, 209, 1705–1719. [CrossRef][PubMed] 84. Lindahl, B.D.; Tunlid, A. Ectomycorrhizal fungi–potential organic matter decomposers, yet not saprotrophs. New Phytol. 2015, 205, 1443–1447. [CrossRef][PubMed] 85. Lauber, C.L.; Strickland, M.S.; Bradford, M.A.; Fierer, N. The influence of soil properties on the structure of bacterial and fungal communities across land-use types. Soil Biol. Biochem. 2008, 40, 2407–2415. [CrossRef] 86. Richard, F.; Moreau, P.A.; Selosse, M.A.; Gardes, M. Diversity and fruiting patterns of ectomycorrhizal and saprobic fungi in an old-growth Mediterranean forest dominated by Quercus ilex L. Can. J. Bot. 2004, 82, 1711–1729. [CrossRef] 87. Azul, A.M.; Castro, P.; Sousa, J.P.; Freitas, H. Diversity and fruiting patterns of ectomycorrhizal and saprobic fungi as indicators of land-use severity in managed woodlands dominated by Quercus suber-a case study from southern Portugal. Can. J. For. Res. 2009, 39, 2404–2417. [CrossRef] 88. Persha, L.; Agrawal, A.; Chhatre, A. Social and ecological synergy: local rulemaking, forest livelihoods, and biodiversity conservation. Science 2011, 331, 1606–1608. [CrossRef][PubMed] 89. Fierer, N.; Nemergut, D.; Knight, R.; Craine, J.M. Changes through time: Integrating microorganisms into the study of succession. Res. Microbiol. 2010, 161, 635–642. [CrossRef][PubMed]

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).